Szewczak Lara B Weinstein
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
Methods Mol Biol. 2008;488:153-66. doi: 10.1007/978-1-60327-475-3_10.
Multicomponent RNA-protein complexes are essential for eukaryotic gene expression. Some, like the spliceosome, have been studied successfully in vitro using biochemical and structural approaches, but many have not been reconstituted in cell-free systems. Nucleotide analog interference mapping (NAIM) can report detailed atomic information about requirements for ribonucleoprotein particle assembly and function in living cells, providing a method to study complexes in a cellular context at a level of detail comparable to many biochemical assays. The method relies on incorporation of phosphorothioate-tagged nucleotide analogs during in vitro transcription, followed by a selection for the active population of molecules and analysis of the selected RNA sequence composition. Xenopus oocytes provide a cellular environment for selecting active molecules based on particle assembly or function. Functional group analysis of complexes assembled in vivo provides predictive models for further investigation either in vivo or in vitro as well as benchmarks for evaluating and refining biochemical and structural models.
多组分RNA-蛋白质复合物对于真核基因表达至关重要。一些复合物，如剪接体，已通过生化和结构方法在体外成功进行了研究，但许多复合物尚未在无细胞系统中重建。核苷酸类似物干扰图谱（NAIM）可以报告有关核糖核蛋白颗粒组装和在活细胞中功能要求的详细原子信息，提供一种在细胞环境中以与许多生化分析相当的详细程度研究复合物的方法。该方法依赖于在体外转录过程中掺入硫代磷酸酯标记的核苷酸类似物，随后选择活性分子群体并分析所选RNA的序列组成。非洲爪蟾卵母细胞提供了一个基于颗粒组装或功能选择活性分子的细胞环境。对体内组装的复合物进行官能团分析可为体内或体外的进一步研究提供预测模型，以及评估和完善生化和结构模型的基准。
